The present invention relates to polymer synthesis. More particularly, the present invention is an improved apparatus for the robotic synthesis of polymers.
DNA synthesis is presently performed on automated instruments which are capable of concurrently producing multiple DNA segments. Frequently the apparatus uses reaction columns in which a support material for the reaction is positioned within the columns on top of inert, porous filters, referred to as frits. The support material generally has a starter material bound to the support onto which desired oligonucleotides may be synthesized. The reaction columns are placed within the automated apparatus and chemicals are added to the columns in sequence in appropriate amounts in an automated fashion. In order to address today""s large demand for high throughput oligosynthesis, most automated apparatuses have a large footprint and take up a great deal of premium laboratory space.
Most currently known automated synthesizers can produce only a few oligonucleotides at a time, which is limited by the number of reaction columns located within the machines. The number of reaction columns is limited as a practical matter by the increased complexity of the plumbing and valving network, as the number of columns increases. In addition, the system must be air tight to avoid contaminating the chemicals with air or water and to avoid human exposure to the chemicals.
U.S. Pat. No. 5,368,823 issued Nov. 29, 1994, and U.S. Pat. No. 5,541,314 issued Jul. 30, 1996, address the need for producing a large number of oligonucleotides by disclosing a method and apparatus for oligonucleotide synthesis in which the plumbing and valving network is simplified. The patents disclose a system in which there is one supply line and one outlet located in the synthesis chamber for the delivery of reagents into the reaction columns. The outlet can be positioned above the inlet end of each of the columns so that nucleotide reagents, capping reagents, deblocking reagents, wash chemicals, etc. can be provided to each of the reaction columns. All of the reagents are located in a supply system which includes reservoirs and valving to connect the reservoirs with the supply line. A flush/prime column is also located within the chamber so that the supply line can be flushed and primed between each different chemical reagent addition. A vacuum source, located outside of the reaction chamber, is connected to the outlet end of the reaction columns to rapidly draw the chemicals from all columns simultaneously, thus leaving the columns dry and ready to receive the next reagent.
The disclosed apparatus in these two patents provides multiple reaction columns, but the single supply line requires flushing and priming between the addition of each reagent. These steps are time consuming and waste reagents. Moreover, a large footprint is required to accommodate a reaction chamber encompassing a) the moving supply line and b) the reaction chambers as well as a vacuum source outside of the reaction chamber. The large footprint is a drawback to space-constrained laboratories.
Another group of patents, U. S. Pat. No. 5,472,672 issued Dec. 5, 1995, U.S. Pat. No. 5,529,756 issued Jun. 25, 1996, and U.S. Pat. No. 5,837,858 issued Nov. 17, 1998, addresses the need for high throughput oligosynthesis by disclosing a polymer synthesis apparatus with many stationary supply lines. The patents disclose an apparatus with a head assembly with many nozzles, with each nozzle coupled to a reagent reservoir. Further, a base assembly has at least one reaction well but can utilize 96-well and other plates. A transport mechanism is coupled to the head assembly and/or base assembly to produce relative movement between the two. The transport mechanism moves horizontally to align a selected reaction well and a selected nozzle to deposit a selected liquid reagent into the reaction well for synthesis of a polymer chain. A sliding seal is positioned between the head assembly and the base assembly to form a common chamber that encloses both the reaction wells and nozzles therein. The seal is constantly being rubbed down by the movement of the metal piece back and forth to move the synthesis block. This wearing down of the seal results in a less efficient seal.
What is needed in modern biological research is a robust, compact and efficient high throughput system for the synthesis of polymers, specifically oligonucleotides.
A polymer synthesizer is disclosed which has a base on which sits a synthesis case, a synthesis block, a means of moving the synthesis block and supports for a reagent shelf. The synthesis case has a loading station, drain station, and water-tolerant and water-sensitive reagent filling stations. The synthesis case has a cover, a first and a second side, a first and a second end, and a bottom side, which contacts the base. The bottom side of the synthesis case has a top face in which there are tracks for the synthesis block. A synthesis block moves back and forth inside the synthesis case and has a top face and an opening in the top face for a synthesis plate with a plurality of wells. The synthesis block also has a collection area under the synthesis plate to drain spent reagents and to optionally accommodate a sample tray. The polymer synthesizer also has a means of moving the synthesis block back and forth in the synthesis case. The load station has a sealable opening through which a multiwell plate is inserted into the synthesis block. The reagent shelf is connected to the upper ends of the supports, which are capable of supporting a plurality of reagent, containers, each reagent container having a tube connecting to a gas source which is used to expel a controlled amount of reagent from the container. The reagent container also dispenses reagent through a tube whose other end connects to a valve that has additional tubes connected to multi-channel manifolds, which in turn have tubes connecting to nozzle blocks at the aqueous and non-aqueous filling stations. The valves are actuated by the computer to dispense fluid to desired wells in the multiwell plate. The polymer synthesizer also has a means of draining the liquid from the synthesis plate. The means for moving the synthesis block can include a pulley, cable and motor.
The means for draining liquid from the synthesis plate can include a pressurized gas source, a pressurized gas inlet on the synthesis case, a pressure plate, a support block, a diaphragm which forms a seal between the top plate and one side of the pressure plate and the support block, a motive means connected to the pressure plate and capable of moving the pressure plate up, and at least one sealing gasket to contact and form a seal with the synthesis plate. The gas enters through the pressurized gas inlet and presses down the diaphragm, which in turn lowers the pressure plate and gasket to form a seal over the synthesis block and increases pressure above the wells, which expels the liquid contents of the wells. The motive means for returning the pressure plate to its original position can be a set of springs.
An automated method of draining synthesis wells in a polymer synthesizer includes the steps of providing the drain station disclosed above, supplying pressurized gas at the pressurized gas inlet, increasing a distensible space between the pressure plate and the diaphragm, pressing down the diaphragm for contact of the gaskets on the pressure plate with the multiwell plate, creating a seal with the multiwell plate, compressing the gasket and the space at each well""s inlet, and expelling liquid or reagents present in the well from the well outlets.
In another embodiment, there is disclosed an automated method for synthesizing polymers, specifically oligonucleotides.
A further understanding of the nature and advantages of the inventions herein may be realized by reference to the remaining portions of the specification and the attached drawings.